The issue of hepatitis B virus (HBV) mutations possibly leading to a gender disparity in the progression of liver diseases has not been explored. We aimed to elucidate the relationships of the novel pre-S1 mutations, W4P/R, with the progression of liver diseases and male predominance in a South Korean chronic cohort by use of a molecular epidemiologic study. We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (RT-PCR) assay for the detection of the W4P/R mutations and applied it to 292 chronic HBV patients. The pre-S1 mutations from 247 (84.6%) of a total of 292 patients were detected by this assay. W4P/R mutants were found to be significantly related to severe liver diseases (hepatocellular carcinoma [HCC] and liver cirrhosis, 12.4% [19/153] of patients, versus chronic hepatitis and asymptomatic carriage, 1.1% [1/94] of patients) (P < 0.001). All of the W4P/R mutants were found in males only. The novel HBV pre-S1 mutations, W4P/R, may be associated with disease severity in male patients chronically infected with HBV genotype C. The W4P/R mutations may provide in part an explanation for the relatively high ratio of male to female incidence in HCC generation in South Korean chronic HBV patients.